Analysis of Molecular Mechanism of ARF-dependent apoptotic cell death

人类肿瘤抑制因子p19ARF、p14ARF由Ink4a/ARF位点编码（Quelle, d.e.et al., (1995) Cell 83,993 -100），在多种癌症中发生突变、缺失或沉默。pl9ARF通过拮抗p53负调节因子Mdm2的活性诱导生长停滞，从而允许p53转录应答(Kamijo， T. et al., (1997) Cell 91, 649-659；Kamijo T. et al., (1998) Proc. Natl。学会科学。《美国法典》95,8292 -829页)。虽然单独激活ARF和稳定p53不足以诱导所有细胞类型的凋亡，但以这种方式上调p53可使细胞在基因毒性应激或癌基因本身诱导的额外凋亡信号下强烈致敏死亡。在本项目中，我们首先分析了ARF依赖性凋亡的机制，并证明了ARF在p53野生型、ARF/p16缺失的细胞中诱导线粒体依赖性凋亡。我们还发现，ARF诱导线粒体释放细胞色素c，降低线粒体膜电位，激活前caspase-9诱导细胞凋亡。我们的研究结果表明，这种凋亡细胞调节是通过线粒体片段中促凋亡Bc1-2家族蛋白Bax和Bim的上调和抗凋亡Bcl-2的下调来实现的。此外，ARF似乎通过p53依赖的方式下调bcl -2，而通过p53不依赖的途径上调Bax/Bim （Nakazawa, Kamuo, Koike, K . Noda, Noda）。2003)最近的一项研究表明，与单独缺乏任何一种基因的小鼠相比，缺乏ARF、p53和MDM2的三敲除（TKO）小鼠每只发生多发且更具侵袭性的肿瘤（Weber， J. D等，（1999）Nat. Cell Biol. 1,20 -26），表明ARF可能通过不依赖于p53/MDM2的方式抑制肿瘤进展。此外，先前的报道表明，小鼠和人的ARF通过nh2末端结构域与MDM2相互作用，但迄今尚未确定ARF的功能结构域和p53/MDM2非依赖性肿瘤抑制的分子机制。综合这些发现，我们接下来利用p53/ rb失活细胞系研究了ARF以p53不依赖的方式诱导凋亡细胞死亡的分子机制，并令人惊讶地发现，ARF的cooh末端具有诱导p53不依赖的细胞死亡的能力。这是第一个描述p19ARF cooh末端凋亡细胞死亡功能的报道。此外，我们还研究了p19ARF cooh末端诱导细胞凋亡的caspase级联以及激活的caspase与控制p19ARF cooh末端诱导细胞凋亡的MAPKs之间的关系。为了进一步研究arf诱导的细胞死亡，我们通过将SV4OT引入arf无效的mef中制备p53/ rb失活细胞。在p53/ rb失活细胞中重新引入ARF可上调线粒体依赖性凋亡细胞死亡，并伴随线粒体中细胞色素c的释放和内在通路caspase的激活，即caspase-9、-3和7。有趣的是，ARF在ARF诱导的细胞中结合SV4OT， p53的再激活似乎是由ARF使SV4OT失活介导的，从而导致线粒体功能障碍（Nakazawa Y和Kamijo T，提交）。少

The tumor suppressor p19ARF, p14ARF in humans, is encoded by the Ink4a/ARF locus (Quelle, D. E.et al., (1995) Cell 83, 993-100) and mutated, deleted, or silenced in many forms of cancer. pl9ARF induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby permitting a p53 transcriptional response (Kamijo, T. et al., (1997) Cell 91, 649-659; Kamijo T. et al., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8292-829'). Although ARF activation and p53 stabilization alone are not sufficient to induce apoptosis in all cell types, upregulation ofp53 in this way strongly sensitizes cells to die in response to genotoxic stresses or additional apoptotic signals induced by oncogenes themselves. In this project, we first analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytocbrome c release from mitochondria, decreases mitochondria mem … More brane potential and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the pro-apoptotic Bc1-2 family proteins Bax and Bim, and down-regulation of anti apopt3tic Bcl-2, in mitochondrial fractions. Additionally, ARF seems to down-tegulate Bc1-2 in a p53-dependent manner, while up-regulating Bax/Bim via a p53-independent pathway (Nakazawa Y, Kamuo, Koike K, Noda T. J Biol Chem. 278(30) : 27888-95. 2003)A recent study showed that triple knockout (TKO) mice lacking ARF, p53, and MDM2 develop multiple and more aggressive tumors per animal than mice lacking either gene alone (Weber, J. D et al., (1999) Nat. Cell Biol. 1, 20-26), indicating that ARF may suppress tumor progression by a p53/MDM2-independent manner. Moreover, previous reports presented that murine and human ARFs interact with MDM2 via NH2-terminal domains, but the functional domain of ARF and the molecular mechanism of p53/MDM2-independent tumor suppression has thus far not been identified. Taken together these findings, we next addressed the molecular mechanism of ARF-induced apoptotic cell death in a p53-independent manner using a p53/Rb-inactivated cell line, and, surprisingly, found that the COOH-terminal of ARF has the ability to induce p53-independent cell death. This is the first report that describes the apoptotic cell death-function of the p19ARF COOH-terminal. Furthermore, we address the responsible caspase cascade for p19ARF COOH-terminal induced apoptosis and relationship between the activated caspases and MAPKs controlling p19ARF COOH-terminal-induced apoptosis. For further investigation of ARF-induced cell death, we made p53/Rb-inactivated cells by introducing SV4OT into ARF-null MEFs. Re-introduction of ARF into the p53/Rb-inactivated cells up-regulated mitochondria-dependent apoptotic cell death accompanied cytochrome c release from mitochondria and activation of the intrinsic pathway caspases, i.e. caspase-9,-3 and-7. Interestingly, ARF bound SV4OT in the ARF-induced cells and re-activation of p53 was seemed to be mediated by the SV4OT inactivation by ARF, resulting in the mitochondrial dysfunction (Nakazawa Y and Kamijo T, submitted). Less